Cycloalkenyl substituted compounds

ABSTRACT

Novel pyridyl or pyrimidinyl substituted cycloalkenyl compounds and compositions for use in therapy

This application is the §371 national stage entry of PCT/US98/21189,filed Oct. 8, 1998, and which claims the benefit of provisionalapplication No. 60/061,351, filed Oct. 8, 1997.

FIELD OF THE INVENTION

This invention relates to novel cycloalkenyl substituted compounds,processes for the preparation thereof, the use thereof in treatingcytokine mediated diseases and pharmaceutical compositions for use insuch therapy.

BACKGROUND OF THE INVENTION

Intracellular signal transduction is the means by which cells respond toextracellular stimuli. Regardless of the nature of the cell surfacereceptor (e. g. protein tyrosine kinase or seven-transmembrane G-proteincoupled), protein kinases and phosphatases along with phopholipases arethe essential machinery by which the signal is further transmittedwithin the cell [Marshall, J. C. Cell, 80, 179-278 (1995)]. Proteinkinases can be categorized into five classes with the two major classesbeing, tyrosine kinases and serine/threonine kinases depending uponwhether the enzyme phosphorylates its substrate(s) on specifictyrosine(s) or serine/threonine(s) residues [Hunter, T., Methods inEnzyrnology (Protein Kinase Classification) p. 3, Hunter, T.; Sefton, B.M.; eds. vol. 200, Academic Press; San Diego, 1991].

For most biological responses, multiple intracellular kinases areinvolved and an individual kinase can be involved in more than onesignaling event. These kinases are often cytosolic and can translocateto the nucleus or the ribosomes where they can affect transcriptionaland translational events, respectively. The involvement of kinases intranscriptional control is presently much better understood than theireffect on translation as illustrated by the studies on growth factorinduced signal transduction involving MAP/ERK kinase [Marshall, C. J.Cell, 80, 179 (1995); Herskowitz, I. Cell, 80, 187 (1995); Hunter, T.Cell, 80, 225 (1995);Seger, R., and Krebs, E. G. FASEB J., 726-735(1995)].

While many signaling pathways are part of cell homeostasis, numerouscytokines (e.g., IL-1 and TNF) and certain other mediators ofinflammation (e.g., COX-2, and iNOS) are produced only as a response tostress signals such as bacterial lippopolysaccharide (LPS). The firstindications suggesting that the signal transduction pathway leading toLPS-induced cytokine biosynthesis involved protein kinases came fromstudies of Weinstein [Weinstein, et al., J. Immunol. 151, 3829(1993)]but the specific protein kinases involved were not identified. Workingfrom a similar perspective, Han [Han, et al., Science 265, 808(1994)]identified murine p38 as a kinase which is tyrosine phosphorylated inresponse to LPS. Definitive proof of the involvement of the p38 kinasein LPS-stimulated signal transduction pathway leading to the initiationof proinflammatory cytokine biosynthesis was provided by the independentdiscovery of p38 kinase by Lee [Lee; et al., Nature, 372, 739(1994)] asthe molecular target for a novel class of anti-inflammatory agents. Thediscovery of p38 (termed by Lee as CSBP 1 and 2) provided a mechanism ofaction of a class of anti-inflammatory compounds for which SK&F 86002was the prototypic example. These compounds inhibited IL-1 and TNFsynthesis in human monocytes at concentrations in the low uM range [Lee,et al., Int. J. Immunopharmac. 10(7), 835(1988)] and exhibited activityin animal models which are refractory to cyclooxygenase inhibitors [Lee;et al., Annals N. Y. Acad. Sci., 696, 149(1993)].

It is now firmly established that CSBP/p38 is a one of several kinasesinvolved in a stress-response signal transduction pathway which isparallel to and largely independent of the analogous mitogen-activatedprotein kinase (MAP) kinase cascade (FIG. 1). Stress signals, includingLPS, pro-inflammatory cytokines, oxidants, UV light and osmotic stress,activate kinases upstream from CSBP/p38 which in turn phosphorylateCSBP/p38 at threonine 180 and tyrosine 182 resulting in CSBP/p38activation. MAPKAP kinase-2 and MAPKAP kinase-3 have been identified asdownstream substrates of CSBP/p38 which in turn phosphorylate heat shockprotein Hsp 27 (FIG. 2). It is not yet known whether MAPKAP-2, MAPKAP-3,Mnk1 or Mnk2 are involved in cytokine biosynthesis or alternatively thatinhibitors of CSBP/p38 kinase might regulate cytokine biosynthesis byblocking a yet unidentified substrate downstream from CSBP/p38 [Cohen,P. Trends Cell Biol., 353-361(1997)].

What is known, however, is that in addition to inhibiting IL-1 and TNF,CSBP/p38 kinase inhibitors (SK&F 86002 and SB 203580) also decrease thesynthesis of a wide variety of pro-inflammatory proteins including,IL-6, IL-8, GM-CSF and COX-2. Inhibitors of CSBP/p38 kinase have alsobeen shown to suppress the TNF-induced expression of VCAM-1 onendothelial cells, the TNF-induced phosphorylation and activation ofcytosolic PLA2 and the IL-1-stimulated synthesis of collagenase andstromelysin. These and additional data demonstrate that CSBP/p38 isinvolved not only cytokine synthesis, but also in cytokine signaling[CSBP/P38 kinase reviewed in Cohen, P. Trends Cell Biol.,353-361(1997)].

Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) are biologicalsubstances produced by a variety of cells, such as monocytes ormacrophages. IL-1 has been demonstrated to mediate a variety ofbiological activities thought to be important in immunoregulation andother physiological conditions such as inflammation [See, e.g.,Dinarello et al., Rev. Infect. Disease, 6, 51 (1984)]. The myriad ofknown biological activities of IL-1 include the activation of T helpercells, induction of fever, stimulation of prostaglandin or collagenaseproduction, neutrophil chemotaxis, induction of acute phase proteins andthe suppression of plasma iron levels.

There are many disease states in which excessive or unregulated IL-1production is implicated in exacerbating and/or causing the disease.These include rheumatoid arthritis, osteoarthritis, endotoxemia and/ortoxic shock syndrome, other acute or chronic inflammatory disease statessuch as the inflammatory reaction induced by endotoxin or inflammatorybowel disease; tuberculosis, atherosclerosis, muscle degeneration,cachexia, psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis,gout, traumatic arthritis, rubella arthritis, and acute synovitis.Recent evidence also links IL-1 activity to diabetes and pancreatic βcells [review of the biological activities which have been attributed toIL-1 Dinarello, J. Clinical Immunology, 5 (5), 287-297 (1985)].

Excessive or unregulated TNF production has been implicated in mediatingor exacerbating a number of diseases including rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions; sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusioninjury, graft vs. host reaction, allograft rejections, fever andmyalgias due to infection, such as influenza, cachexia secondary toinfection or malignancy, cachexia, secondary to acquired immunedeficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloidformation, scar tissue formation, Crohn's disease, ulcerative colitis,or pyresis.

Interleukin-8 (IL-8) is a chemotactic factor produced by several celltypes including mononuclear cells, fibroblasts, endothelial cells, andkeratinocytes. Its production from endothelial cells is induced by IL-1,TNF, or lipopolysachharide (LPS). IL-8 stimulates a number of functionsin vitro. It has been shown to have chemoattractant properties forneutrophils, T-lymphocytes, and basophils. In addition it induceshistamine release from basophils from both normal and atopic individualsas well as lysozomal enzyme release and respiratory burst fromneutrophils. IL-8 has also been shown to increase the surface expressionof Mac-1 (CD11b/CD18) on neutrophils without de novo protein synthesis,this may contribute to increased adhesion of the neutrophils to vascularendothelial cells. Many diseases are characterized by massive neutrophilinfiltration. Conditions associated with an increased in IL-8 production(which is responsible for chemotaxis of neutrophil into the inflammatorysite) would benefit by compounds which are suppressive of IL-8production.

IL-1 and TNF affect a wide variety of cells and tissues and thesecytokines as well as other leukocyte derived cytokines are important andcritical inflammatory mediators of a wide variety of disease states andconditions. The inhibition of these cytokines is of benefit incontrolling, reducing and alleviating many of these disease states.

Inhibition of signal transduction via CSBP/p38, which in addition toIL-1, TNF and IL-8 described above is also required for the synthesisand/or action of several additional pro-inflammatory proteins (i.e.,IL-6, GM-CSF, COX-2, collagenase and stromelysin), is expected to be ahighly effective mechanism for regulating the excessive and destructiveactivation of the immune system. This expectation is supported by thepotent and diverse anti-inflammatory activities described for CSBP/p38kinase inhibitors [Badger, et al., J. Pharm. Exp. Thera 279 (3):1453-1461.(1996); Griswold, et al, Pharmacol. Comm. 7, 323-229 (1996)].

There remains a need for treatment, in this field, for compounds whichare cytokine suppressive anti-inflammatory drugs, i.e. compounds whichare capable of inhibiting the CSBP/p38/RK kinase.

SUMMARY OF THE INVENTION

This invention relates to the novel compounds of Formula (I), andpharmaceutical compositions comprising a compound of Formula (I), and apharmaceutically acceptable diluent or carrier.

This invention relates to a method of treating a CSBP/RK/p38 kinasemediated disease, in a mammal in need thereof, which comprisesadministering to said mammal an effective amount of a compound ofFormula (I).

This invention also relates to a method of inhibiting cytokines and thetreatment of a cytokine mediated disease, in a mammal in need thereof,which comprises administering to said mammal an effective amount of acompound of Formula (I).

This invention more specifically relates to a method of inhibiting theproduction of IL-1 in a mammal in need thereof which comprisesadministering to said mammal an effective amount of a compound ofFormula (I).

This invention more specifically relates to a method of inhibiting theproduction of IL-8 in a mammal in need thereof which comprisesadministering to aid mammal an effective amount of a compound of Formula(I).

This invention more specifically relates to a method of inhibiting theproduction of TNF in a mammal in need thereof which comprisesadministering to said mammal an effective amount of a compound ofFormula (I).

Accordingly, the present invention provides for a compound of theformula:

wherein

R₁ is 4-pyridyl, 4-pyrimidinyl, 4-pyridazinyl, 1,2,4-triazin-5-yl,4-quinolyl, 6-isoquinolinyl, quinazolin-4-yl, 1-imidazolyl or1-benzimidazolyl ring, which ring is optionally substitutedindependently one to three times with Y, NHR_(a), optionally substitutedC₁₋₄ alkyl, halogen, hydroxyl, optionally substituted C₁₋₄ alkoxy,optionally substituted C₁₋₄ alkylthio, optionally substituted C₁₋₄alkylsulfinyl, CH₂OR₁₂, amino, mono and di- C₁₋₆ alkyl substitutedamino, N(R₁₀)C(O)R_(b), N(R₁₀)S(O)₂R_(d), or an N-heterocyclyl ringwhich ring has from 5 to 7 members and optionally contains an additionalheteroatom selected from oxygen, sulfur or NR₁₅;

Y is X₁—R_(a);

X₁ is sulfur or oxygen;

R_(a) is C₁₋₆ alkyl, aryl, arylC₁₋₆ alkyl, heterocyclic,heterocyclylC₁₋₆ alkyl, heteroaryl, or heteroarylC₁₋₆ alkyl moiety,wherein each of these moieties may be optionally substituted;

R_(b) is hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl,heteroaryl, heteroarylC₁₋₄ alkyl, heterocyclyl, or heterocyclylC1-4alkyl;

R_(d) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl,heteroarylC₁₋₄ alkyl, heterocyclyl, or heterocyclylC₁₋₄ alkyl;

Rg is independently hydrogen or R₂;

n is 0, or an integer having a value of 1 to 10;

v is 0, or an integer having a value of 1 or 2;

m is 0, or the integer having a value of 1 or 2;

m′ is an integer having a value of 1 or 2,

m″ is 0, or an integer having a value of 1 to 5;

s is an integer having a value of 1 to 5;

t is 0, or an integer having a value of 1, 2 or 3;

R₂ is independently hydrogen, (CR₁₀R₂₃)_(n)OR₉, (CR₁₀R₂₃)_(n)OR₁₁, C₁₋₁₀alkyl, halo-substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₇cycloalkyl, C₃₋₇cycloalkylC₁₋₁₀ alkyl, C₅₋₇ cycloalkenyl, C₅₋₇cycloalkenyl C₁₋₁₀ alkyl, aryl, arylC₁₋₁₀ alkyl, heteroaryl,heteroarylC₁₋₁₀ alkyl, heterocyclyl, heterocyclylC₁₋₁₀ alkyl,(CR₁₀R₂₃)_(n)S(O)_(m)R₁₈, (CR₁₀R₂₃)_(n)NHS(O)₂R₁₈, (CR₁₀R₂₃)_(n)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)NO₂, (CR₁₀R₂₃)_(n)CN, (CR₁₀R₂₃)_(n)S(O)m′NR₁₃R₁₄,(CR₁₀R₂₃)_(n)C(Z)R₁₁, (CR₁₀R₂₃)_(n)OC(Z)R₁₁(CR₁₀R₂₃)_(n)C(Z)OR₁₁,(CR₁₀R₂₃)_(n)C(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)C(Z)NR₁₁OR₉,(CR₁₀R₂₃)_(n)NR₁₀C(Z)R₁₁, (CR₁₀R₂₃)_(n)NR₁₀C(Z)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)N(OR₆)C(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)N(OR6)C(Z)R₁₁,(CR₁₀R₂₃)_(n)C(═NOR₆)R₁₁, (CR₁₀R₂₃)_(n)NR₁₀C(═NR19)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)OC(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)NR₁₀C(Z)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)NR₁₀C(Z)OR₁₀, 5-(R₁₈)-1,2,4-oxadizaol-3-yl or4-(R₁₂)-5-(R₁₈R₁₉)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein thecycloalkyl, cycloalkyl alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic and heterocyclic alkyl may be optionallysubstituted;

R₃ is heterocyclyl, heterocyclylC₁₋₁₀ alkyl or R₈;

R₄ is phenyl, naphth-1-yl or naphth-2-yl, or heteroaryl, which isoptionally substituted by one to three substituents, each of which isindependently selected, and which, for a 4-phenyl, 4-naphth-1-yl,5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro,C(Z)NR₇R₁₇, C(Z)OR₁₆, (CR₁₀R₂₀)_(v)COR₁₂, SR₅, S(O)R₅, OR₁₂,halo-substituted-C₁₋₄ alkyl, C₁₋₄alkyl, ZC(Z)R₁₂, NR₁₀C(Z)R₁₆, or(CR₁₀R₂₀)vNR₁₀R₂₀ and which, for other positions of substitution, ishalogen, cyano, nitro, phenyl, C(Z)NR₁₃R₁₄, C(Z)OR₃, (CR₁₀R₂₀)_(m″)COR₃,S(O)_(m)R₃, OR₃, halosubstituted-C₁₋₄ alkyl, C₁₋₁₀ alkyl, ZC(Z)R₃,optionally substituted phenyl, (CR₁₀R₂₀)_(m″)NR₁₀C(Z)R₃,NR₁₀S(O)_(m′)R₈, NR₁₀S(O)_(m′)NR₇R₁₇, or (CR₁₀R₂₀)_(m″)NR₁₃R₁₄;

R₅ is hydrogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl or NR₇R₁₇,excluding the moieties SR₅ being SNR₇R₁₇ and SOR₅ being SOH;

R₆ is hydrogen, a pharmaceutically acceptable cation, C₁₋₁₀ alkyl, C₃₋₇cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl, heteroarylC₁₋₄ alkyl,heterocyclic, aroyl, or C₁₋₁₀ alkanoyl;

R₇ and R₁₇ is each independently selected from hydrogen or C₁₋₄ alkyl orR₇ and R₁₇ together with the nitrogen to which they are attached form aheterocyclic ring of 5 to 7 members which ring optionally contains anadditional heteroatom selected from oxygen, sulur or NR₁₅;

R₈ is C₁₋₁₀ alkyl, halo-substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₇ cycloalkyl, C₅₋₇ cycloalkenyl, aryl, arylC₁₋₁₀ alkyl,heteroaryl, heteroarylC₁₋₁₀ alkyl, (CR₁₀R₂₀)_(n)OR₁₁,(CR₁₀R₂₀)_(n)S(O)_(m)R₁₈, (CR₁₀R₂₀)_(n)NHS(O)₂R₁₈, or(CR₁₀R₂₀)_(n)NR₁₃R₁₄; wherein the aryl, arylalkyl, heteroaryl,heteroaryl alkyl may be optionally substituted;

R₉ is hydrogen, C(Z)R₁₁ or optionally substituted C₁₋₁₀ alkyl, S(O)₂R₁₈,optionally substituted aryl or optionally substituted arylC₁₋₄ alkyl;

R₁₀ and R₂₀ is each independently selected from hydrogen or C₁₋₄ alkyl;

R₁₁ is hydrogen, C₁₋₁₀ alkyl, C₃₋₇ cycloalkyl, heterocyclyl,heterocyclyl C₁₋₁₀ alkyl, aryl, arylC₁₋₁₀ alkyl, heteroaryl or aheteroarylC₁₋₁₀ alkyl moiety, wherein the aryl, arylalkyl, heteroaryl,heteroaryl alkyl, heterocyclyl or heterocyclylalkyl moieties may beoptionally substituted;

R₁₂ is hydrogen or R₁₆;

R₁₃ and R₁₄ is each independently selected from hydrogen or optionallysubstituted C₁₋₄ alkyl, optionally substituted aryl or optionallysubstituted aryl-C₁₋₄alkyl, or together with the nitrogen which they areattached form a heterocyclic ring of 5 to 7 members which ringoptionally contains an additional heteroatom selected from oxygen,sulfur or NR₉;

R₁₅ is hydrogen, C₁₋₄ alkyl or C(Z)—C₁₋₄ alkyl;

R₁₆ is C₁₋₄ alkyl, halo-substituted-C₁₋₄ alkyl, or C₃₋₇ cycloalkyl;

R₁₈ is C₁₋₁₀ alkyl, C₃₋₇ cycloalkyl, heterocyclyl, aryl, arylC₁₋₁₀alkyl, heterocyclyl, heterocyclyl-C₁₋₁₀ alkyl, heteroaryl or aheteroarylalkyl moiety, wherein the aryl, arylalkyl, heteroaryl,heteroaryl alkyl, heterocyclyl or heterocyclylalkyl moieties may beoptionally substituted;

R₁₉ is hydrogen, cyano, C₁₋₄ alkyl, C₃₋₇ cycloalkyl or aryl;

R₂₃ is hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl,heteroaryl, heteroarylC₁₋₄ alkyl, heterocyclyl, or heterocyclylC₁₋₄alkyl moiety, all of which moieties may be optionally substituted;

Z is oxygen or sulfur;

or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The novel compounds of Formula (I) may also be used in association withthe veterinary treatment of mammals, other than humans, in need ofinhibition of cytokine inhibition or production. In particular, cytokinemediated diseases for treatment, therapeutically or prophylactically, inanimals include disease states such as those noted herein in the Methodsof Treatment section, but in particular viral infections. Examples ofsuch viruses include, but are not limited to, lentivirus infections suchas, equine infectious anaemia virus, caprine arthritis virus, visnavirus, or maedi virus or retrovirus infections, such as but not limitedto feline immunodeficiency virus (FIV), bovine immunodeficiency virus,or canine immunodeficiency virus or other retroviral infections.

In Formula (I), suitable R₁ moieties includes 4-pyridyl, 4-pyrimidinyl,4-pyridazinyl, 1,2,4-triazin-5-yl, 4-quinolyl, 6-isoquinolinyl,4-quinazolinyl, 1-iraidazolyl and 1-benzimidazolyl, of which the4-pyridyl, 4-pyrimidinyl and 4-quinolyl are preferred. More preferred isa substituted 4-pyrimidinyl or substituted 4-pyridyl moiety, and mostpreferred is a substituted 4-pyrimidinyl ring. A preferred ringplacement of the R₁ substituent on the 4-pyridyl derivative is the2-position, such as 2-methoxy-4-pyridyl. A preferred ring placement onthe 4-pyrimidinyl ring is also at the 2-position, such as in2-methoxy-pyrimidinyl.

The R₁ moiety may be optionally substituted one or more times,independently, by suitable substituents. Such substituents include Y,NHR_(a), optionally substituted C₁₋₄ alkyl, halogen, hydroxyl,optionally substituted C₁₋₄ alkoxy, optionally substituted C₁₋₄alkylthio, optionally substituted C₁₋₄ alkylsulfinyl, CH₂OR₁₂, amino,mono and di-C₁₋₆ alkyl substituted amino, N(R₁₀)C(O)R_(b);N(R₁₀)S(O)₂R_(d); or an N-heterocyclyl ring which ring has from 5 to 7members and optionally contains an additional heteroatom selected fromoxygen, sulfur or NR₁₅.

Suitably Y is X₁—R_(a); and X₁ is oxygen or sulfur, preferably oxygen.

Suitably, R_(a) is C₁₋₆alkyl, aryl, arylC₁₋₆alkyl, heterocyclic,heterocyclylC₁₋₆ alkyl, heteroaryl, or heteroarylC₁₋₆alkyl, wherein eachof these moieties may be optionallysubstituted as defined herein.

When the substituent is contains the R_(a) moiety, and R_(a) is aryl, itis preferably phenyl or naphthyl. When R_(a) is an aryl alkyl, it ispreferably benzyl or napthylmethyl. When R_(a) is heterocyclic orheterocyclic alkyl moiety, the heterocyclic portion is preferablypyrrolindinyl, piperidine, morpholino, tetrahydropyran,tetrahydrothiopyranyl, tetrahydrothiopyran-sulfinyl,tetrahydrothio-pyransulfonyl, pyrrolindinyl, indole, or piperonyl. It isnoted that the heterocyclic rings herein may contain unsaturation, suchas in an indole ring. When R_(a) is a heteroaryl or heteroarylalkylmoiety it is as defined herein.

These R_(a) aryl, heterocyclic and heteroaryl rings may also beoptionally substituted one or more times independently with halogen;C₁₋₄ alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl;halosubstituted alkyl, such as CF₃; hydroxy; hydroxy substituted C₁₋₄alkyl; C₁₋₄ alkoxy, such as methoxy or ethoxy; S(O)_(m)alkyl and S(O)maryl (wherein m is 0, 1, or 2); C(O)OR₁₁, such as C(O)C₁₋₄ alkyl orC(O)OH moieties; C(O)R₁₁; OC(O)R_(c); O—(CH₂)s—O—, such as in a ketal ordioxyalkylene bridge, and s is 1 to 3; amino; mono- and di-C₁₋₆alkylsubstituted amino, N(R₁₀)C(O)R_(b); N(R₁₀)S(O)₂R_(d); C(O)NR₁₀R₂₀;S(O)₂(CR₁₀R₂₀)_(t)NR₁₃R₁₄ (wherein t is 0, or an integer of 1 to 3);cyano, nitro, or an N-heterocyclyl ring which ring has from 5 to 7members and optionally contains an additional heteroatom selected fromoxygen, sulfur or NR₁₅; aryl, such as phenyl; an optionally substitutedarylalkyl, such as benzyl or phenethyl; aryloxy, such as phenoxy; orarylalkyloxy such as benzyloxy.

Suitably, R_(b) is hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄alkyl, heteroaryl, heteroarylC₁₋₄alkyl, heterocyclyl, orheterocyclylC₁₋₄ alkyl, wherein all of which may be optionallysubstituted.

Suitably, R_(c) is hydrogen, optionally substituted C₁₋₆ alkyl, C₃₋₇cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl, heteroarylC₁₋₄alkyl,heterocyclyl, or heterocyclylC₁₋₄ alkyl moiety, wherein all of which maybe optionally substituted.

Suitably, R_(d) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl,heteroaryl, heteroarylC₁₋₄alkyl, heterocyclyl, or heterocyclylC₁₋₄alkyl, wherein all of which may be optionally substituted.

When the R_(a) moiety is an alkyl group it may be optionally substitutedas defined herein. Also the alkyl portion of the R₁ substituents, themono- and di-C₁₋₆ alkyl amino moieties, may also be halo substituted.

Preferably, the R_(a) group is an alkyl, such as methyl, an optionallysubstituted aryl, such as phenyl, or an optionally substitutedarylalkyl, such as benzyl.

When the R₁ optional substituent group is N(R₁₀)C(O) R_(b), R_(b) ispreferably a C₁₋₆ alkyl; and R₁₀ is preferably hydrogen. It is alsorecognized that all the R_(b) moieties, in particular the C₁₋₆ alkylgroup may be optionally substituted, preferably from one to three timesas defined herein. Preferably R_(b) is C₁₋₆ alkyl substituted withhalogen, such as fluorine, as in trifluoromethyl or trifluroethyl.

Suitably, R₄ is phenyl, naphth-1-yl or naphth-2-yl, or a heteroarylring. Preferably R₄ is a phenyl or naphthyl ring.

Suitable substitutions for R₄ when this is a 4-phenyl, 4-naphth-1-yl,5-naphth-2-yl or 6-naphth-2-yl moiety are one or three, preferably oneto two, substituents each of which are independently selected fromhalogen, SR₅, SOR₅, OR₁₂, CF₃, or (CR₁₀R₂₀)_(v)NR₁₀R₂₀, and for otherpositions of substitution on these rings preferred substitution ishalogen, S(O)_(m)R₃, OR₃, CF₃, (CR₁₀R₂₀)_(m″)NR₁₃R₁₄, NR₁₀C(Z)R₃ andNR₁₀S(O)_(m′)R₈. Preferred substituents for the 4-position in phenyl andnaphth-1-yl and on the 5-position in naphth-2-yl include halogen,especially fluoro and chloro, and SR₅ and SOR₅ wherein R₅ is preferablya C₁₋₂ alkyl, more preferably methyl; of which the fluoro and chloro ismore preferred, and most especially preferred is fluoro. For all othersubstituents, in particular for the 3-position in phenyl and naphth-1-ylrings, the substituents are independently selected from halogen,especially fluoro and chloro; OR₃, especially C₁₋₄ alkoxy; CF₃, NR₁₀R₂₀,such as amino; NR₁₀C(Z)R₃, especially NHCO(C₁₋₁₀ alkyl);NR₁₀S(O)_(m′)R₈, especially NHSO₂(C₁₋₁₀ alkyl); and SR₃ and SOR₃ whereinR₃ is preferably a C₁₋₂ alkyl, more preferably methyl.

When the phenyl ring is disubstituted, preferably it is two independenthalogen moieties, such as fluoro and chloro, preferably di-chioro andmore preferably in the 3, 4-position. It is also preferred that for the3-position of both the OR₃ and ZC(Z)R₃ moieties, that the R₃ may alsoinclude hydrogen.

Preferably, the R₄ moiety is an unsubstituted or substituted phenylmoiety. More preferably, R₄ is phenyl or phenyl substituted at the4-position with fluoro and/or substituted at the 3-position with fluoro,chloro, C₁₋₄ alkoxy, methanesulfonamido or acetamido, or R₄ is a phenyldi-substituted at the 3,4-position independently with chloro or fluoro,more preferably chloro. Most preferably, R₄ is 4-fluorophenyl.

In Formula (I), Z is suitably oxygen or sulfur.

Suitably, R₂ is hydrogen, (CR₁₀R₂₃)_(n)OR₉, (CR₁₀R₂₃)_(n)OR₁₁,C₁₋₁₀alkyl, halo-substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,C₃₋₇ cycloalkyl, C₃₋₇cycloalkylC₁₋₁₀ alkyl, C₅₋₇ cycloalkenyl, C₅₋₇cycloalkenyl C₁₋₁₀alkyl, aryl, arylC₁₋₁₀ alkyl, heteroaryl,heteroarylC₁₋₁₀alkyl, heterocyclyl, heterocyclylC₁₋₁₀ alkyl,(CR₁₀R₂₃)_(n)S(O)_(m)R₁₈, (CR₁₀R₂₃)_(n)NHS(O)₂R₁₈, (CR₁₀R₂₃)_(n)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)NO₂, (CR₁₀R₂₃)_(n)CN, (CR₁₀R₂₃)_(n)S(O)_(m′)NR₁₃R₁₄,(CR₁₀R₂₃₀)_(n)C(Z)R₁₁, (CR₁₀R₂₃)_(n)OC(Z)R₁₁, (CR₁₀R₂₃)_(n)C(Z)OR₁₁,(CR₁₀R₂₃)_(n)C(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)C(Z)NR₁₁OR₉,(CR₁₀R₂₃)_(n)NR₁₀C(Z)R₁₁, (CR₁₀R₂₃)_(n)NR₁₀C(Z)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)N(OR₆)C(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)N(OR₆)C(Z)R₁₁,(CR₁₀R₂₃)_(n)C(═NOR₆)R₁₁, (CR₁₀R₂₃)_(n)NR₁₀C(═NR₁₉)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)OC(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)NR₁₀C(Z)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)NR₁₀C(Z)OR₁₀, 5-(R₁₈)-1,2,4-oxadizaol-3-yl or4-(R₁₂)-5-(R₁₈R₁₉)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein thecycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic and heterocyclic alkyl groups may be optionallysubstituted.

Suitably, R₂₃ is hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄alkyl, heteroaryl, heteroarylC₁₋₄alkyl, heterocyclyl, orheterocyclylC₁₋₄ alkyl moiety, all of which may be optionallysubstituted as defined below.

Preferably, R₂ is hydrogen, an optionally substituted heterocyclyl ring,and optionally substituted heterocyclylC₁₋₁₀ alkyl, an optionallysubstituted C₁₋₁₀ alkyl, an optionally substituted C₃₋₇cycloalkyl, anoptionally substituted C₃₋₇cycloalkyl C₁₋₁₀ alkyl, (CR₁₀R₂₃)_(n)C(Z)OR₁₁group, (CR₁₀R₂₃)_(n)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)NHS(O)₂R₁₈,(CR₁₀R₂₃)_(n)S(O)_(m)R₁₈, an optionally substituted aryl; an optionallysubstituted arylC₁₋₁₀ alkyl, (CR₁₀R₂₃)_(n)OR₁₁, (CR₁₀R₂₃)_(n)C(Z)R₁₁, or(CR₁₀R₂₃)_(n)C(═NOR₆)R₁₁ group.

Preferably, R₂ is selected from hydrogen, C₁₋₁₀ alkyl, optionallysubstituted heterocyclyl, optionally substituted heterocyclylC₁₋₁₀alkyl, (CR₁₀R₂₃)_(n)NS(O)₂R₁₈, (CR₁₀R₂₃)_(n)S(O)_(m)R₁₈, arylC₁₋₁₀alkyl, (CR₁₀R₂₃)_(n)NR₁₃R₁₄, optionally substituted C₃₋₇cycloalkyl, oroptionally substituted C₃₋₇cycloalkyl C₁₋₁₀ alkyl.

When R₂ is an optionally substituted heterocyclyl, the ring ispreferably a morpholino, pyrrolidinyl, or a piperidinyl group. When thering is optionally substituted, the substituents may be directlyattached to the free nitrogen, such as in the piperidinyl group orpyrrole ring, or on the ring itself. Preferably the ring is a piperidineor pyrrole, more preferably piperidine. The heterocyclyl ring may beoptionally substituted one to four times independently by halogen; C₁₋₄alkyl; aryl, such as phenyl; aryl alkyl, such as benzyl—wherein the arylor aryl alkyl moieties themselves may be optionally substituted (as inthe definition section below); C(O)OR₁₁, such as the C(O)C₁₋₄ alkyl orC(O)OH moieties; C(O)H; C(O)C₁₋₄ alkyl, hydroxy substituted C₁₋₄ alkyl,C₁₋₄ alkoxy, S(O)_(m)C₁₋₄ alkyl (wherein m is 0, 1, or 2), NR₁₀R₂₀(wherein R₁₀ and R₂₀ are independently hydrogen or C₁₋₄alkyl).

Preferably if the ring is a piperidine, the substituents are directlyattached on the available nitrogen, i.e, a 1-Formyl-4-piperidine,1-benzyl-4-piperidine, 1-methyl-4-piperidine,1-ethoxycarbonyl-4-piperidine. If the ring is substituted by an alkylgroup and the ring is attached in the 4-position, it is preferablysubstituted in the 2- or 6-position or both, such as2,2,6,6-tetramethyl-4-piperidine.

When R₂ is an optionally substituted heterocyclyl C₁₋₁₀ alkyl group, thering is preferably a morpholino, pyrrolidinyl, or a pipedidinyl group.Preferably this alkyl moiety is from 1 to 4, more preferably 3 or 4, andmost preferably 3, such as in a propyl group. Preferred heterocyclicalkyl groups include but are not limited to, morpholino ethyl,morpholino propyl, pyrrolidinyl propyl, and piperidinyl propyl moieties.

When R₂ is an optionally substituted C₃₋₇cycloalkyl, or an optionallysubstituted C₃₋₇cycloalkyl C₁₋₁₀ alkyl, the cycloalkyl group ispreferably a C₄ or C₆ ring, most preferably a C₆ ring, which ring isoptionally substituted. The cycloalkyl ring may be optionallysubstituted one to three times independently by halogen, such asfluorine, chlorine, bromine or iodine; hydroxy; OC(O)R_(b), C₁₋₁₀alkoxy, such as methoxy or ethoxy; S(O)_(m) alkyl, wherein m is 0, 1, or2, such as methylthio, methylsulfinyl or methylsulfonyl; S(O)_(m)aryl;cyano, nitro, amino, mono & di-substituted amino, such as in the NR₇R₁₇group, wherein R₇ and R₁₇ are as defined in Formula (I), or where theR₇R₁₇ may cyclize together with the nitrogen to which they are attachedto form a 5 to 7 membered ring which optionally includes an additionalheteroatom selected from oxygen, sulfur or NR₁₅; N(R₁₀)C(O)X₁ and X₁ isC₁₋₄ alkyl, aryl or arylC₁₋₄alkyl; C₁₋₁₀ alkyl, such as methyl, ethyl,propyl, isopropyl, or t-butyl; optionally substituted alkyl wherein thesubstituents are halogen, (such as CF₃), hydroxy, nitro, cyano, amino,mono & di-substituted amino, such as in the NR₇R₁₇ group, S(O)m alkyland S(O)m aryl, wherein m is 0, 1 or 2; optionally substituted alkylene,such as ethylene or propylene; optionally substituted alkyne, such asethyne; C(O)OR₁₁, such as the free acid or methyl ester derivative; thegroup R_(e); C(O)H; =O; ═N—OR₁₁; N(H)—OH (or substituted alkyl or arylderivatives thereof on the nitrogen or the oxime moiety);N(OR_(f))—C(O)—R₂₁; an optionally substituted aryl, such as phenyl; anoptionally substituted arylC₁₋₄alkyl, such as benzyl of phenethyl; anoptionally substituted heterocycle or heterocyclic C₁₋₄alkyl, andfurther these aryl, arylalkyl, heterocyclic, and heterocyclic alkylmoieties are optionally substituted one to two times by halogen,hydroxy, C₁₋₁₀ alkoxy, S(O)_(m) alkyl, cyano, nitro, amino, mono &di-substituted amino, such as in the NR₇R₁₇ group, an alkyl, or anhalosubstituted alkyl.

Suitably R_(e) is a 1,3-dioxyalkylene group of the formula—O—(CH₂)_(s)—O—, wherein s is 1 to 3, preferably s is 2 yielding a1,3-dioxyethylene moiety, or ketal functionality.

Suitably R_(f) is hydrogen, a pharmaceutically acceptable cation, aroylor a C₁₋₁₀ alkanoyl group.

Suitably R₂₁ is NR₂₂R₂₄; alkyl₁₋₆; halosubstituted alkyl₁₋₆; hydroxysubstituted alkyl₁₋₆; alkenyl₂₋₆; aryl or heteroaryl optionallysubstituted by halogen, alkyl₁₋₆, halosubstituted alkyl₁₋₆, hydroxyl, oralkoxy₁₋₆.

Suitably R₂₂ is H or alkyl₁₋₆.

Suitably R₂₄ is H, alkyl₁₋₆, aryl, benzyl, heteroaryl, alkyl substitutedby halogen or hydroxyl, or phenyl substituted by a member selected fromthe group consisting of halo, cyano, alkyl₁₋₁₂, alkoxy₁₋₆,halosubstituted alkyl₁₋₆, alkylthio, alkylsulphonyl, or alkylsulfinyl;or R₂₂ and R₂₄ may together with the nitrogen to which they are attachedform a ring having 5 to 7 members, which members may be optionallyreplaced by a heteroatom selected from oxygen, sulfur or nitrogen. Thering may be saturated or contain more than one unsaturated bond.Preferably R₂₁ is NR₂₂R₂₄ and R₂₂ and R₂₄ are preferably hydrogen.

When the R₂ cycloalkyl moiety is substituted by NR₇R₁₇ group, or NR₇R₁₇C₁₋₁₀ alkyl group, and the R₇ and R₁₇ are as defined in Formula (I), thesubstituent is preferably an amino, amino alkyl, or an optionallysubstituted pyrrolidinyl moiety.

A preferred position of ring substitution on the C6 cycloalkyl moiety isthe 4-position. When the cycloalkyl ring is di-substituted it ispreferably di-substituted at the 4 position, such as in:

wherein R^(1′) and R^(2′) are independently the optional substituentsindicated above for R₂. Preferably, R^(1′) and R^(2′) are hydrogen,hydroxy, alkyl, substituted alkyl, optionally substituted alkyne, aryl,arylalkyl, NR₇R₁₇, and N(R₁₀)C(O)R₁₁. Suitably, alkyl is C₁₋₄ alkyl,such as methyl, ethyl, or isopropyl; NR₇R₁₇ and NR₇R₁₇ alkyl, such asamino, methylamino, aminomethyl, aminoethyl; substituted alkyl such asin cyanomethyl, cyanoethyl, nitroethyl, pyrrolidinyl; aryl such as inphenyl; arylalkyl, such as in benzyl; optionally substituted alkyne,such as ethyne or propynyl; or together R^(1′) and R^(2′) are a ketofunctionality.

For compounds of Formula (I), s is 0, or an integer having a value of 1to 5.

For compounds of Formula (I), t is 0 or an integer having a value of 1to 3. This results in a 4 to 7 membered cycloalkenyl ring which isoptionally substituted by the R2 moiety.

For compounds of Formula (I), Rg is independently hydrogen or R₂.

In all instances herein where there is an alkenyl or alkynyl moiety as asubstituent group, the unsaturated linkage, i.e., the vinylene oracetylene linkage is preferably not directly attached to the nitrogen,oxygen or sulfur moieties, for instance in OR₃, or for certain R₂moieties.

Suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of inorganic and organicacids, such as hydrochloric acid, hydrobromic acid, sulphuric acid,phosphoric acid, methane sulphonic acid, ethane sulphonic acid, aceticacid, malic acid, tartaric acid, citric acid, lactic acid, succinicacid, fumaric acid, maleic acid, benzoic acid, salicylic acid,phenylacetic acid and mandelic acid. In addition, pharmaceuticallyacceptable salts of compounds of Formula (I) may also be formed with apharmaceutically acceptable cation, for instance, if a substituent groupcomprises a carboxy moiety. Suitable pharmaceutically acceptable cationsare well known to those skilled in the art and include alkaline,alkaline earth, ammonium and quaternary ammonium cations.

As used herein, “optionally substituted”, unless specifically defined,shall mean such groups as halogen, such as fluorine, chlorine, bromineor iodine; hydroxy; hydroxy substituted C₁₋₁₀alkyl; C₁₋₁₀ alkoxy, suchas methoxy or ethoxy; S(O)m alky), wherein m is 0, 1 or 2, such asmethylthio, methylsulfinyl or methylsulfonyl; halosubstituted C₁₋₁₀alkoxy; amino, mono & di-substituted amino, such as in the NR₇R₁₇ group;or where the R₇R₁₇ may together with the nitrogen to which they areattached cyclize to form a 5 to 7 membered ring which optionallyincludes an additional heteroatom selected from O/N/S; C₁₋₁₀ alkyl,cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl,isopropyl, t-butyl, etc. or cyclopropyl methyl; halosubstituted C₁₋₁₀alkyl, such CF₃; an optionally substituted aryl, such as phenyl, or anoptionally substituted arylalkyl, such as benzyl or phenethyl, whereinthese aryl moieties may also be substituted one to three times byhalogen; hydroxy; hydroxy substituted alkyl; C₁₋₁₀ alkoxy; S(O)_(m)alkyl; amino, mono & di-substituted amino, such as in the NR₇R₁₇ group;alkyl, or CF₃.

The following terms, as used herein, refer to:

“halo” or “halogens”, include the halogens: chloro, fluoro, bromo andiodo.

“C₁₋₁₀alkyl” or “alkyl”—both straight and branched chain radicals of 1to 10 carbon atoms, unless the chain length is otherwise limited,including, but not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl and the like.

The term “cycloalkyl” is used herein to mean cyclic radicals, preferablyof 3 to 8 carbons, including but not limited to cyclopropyl,cyclopentyl, cyclohexyl, and the like.

The term “cycloalkenyl” is used herein to mean cyclic radicals,preferably of 5 to 8 carbons, which have at least one bond including butnot limited to cyclopentenyl, cyclohexenyl, and the like.

The term “alkenyl” is used herein at all occurrences to mean straight orbranched chain radical of 2-10 carbon atoms, unless the chain length islimited thereto, including, but not limited to ethenyl, 1-propenyl,2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like.

“aryl”—phenyl and naphthyl.

“heteroaryl” (on its own or in any combination, such as “heteroaryloxy”,or “heteroaryl alkyl”)—a 5-10 membered aromatic ring system in which oneor more rings contain one or more heteroatoms selected from the groupconsisting of N, O or S, such as, but not limited, to pyrrole, pyrazole,furan, thiophene, indole, quinoline, isoquinoline, quinazolinyl,pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole,imidazole, or benzimidazole.

“heterocyclic” (on its own or in any combination, such as“heterocyclylalkyl”)—a saturated or partially unsaturated 4-10 memberedring system in which one or more rings contain one or more heteroatomsselected from the group consisting of N, O, or S; such as, but notlimited to, pyrrolidine, piperidine, piperazine, morpholine,tetrahydropyran, or imidazolidine.

The term “aralkyl” or “heteroarylalkyl” or “heterocyclicalkyl” is usedherein to mean C₁₋₄ alkyl as defined above attached to an aryl,heteroaryl or heterocyclic moiety as also defined herein unlessotherwise indicate.

“sulfinyl”—the oxide S(O) of the corresponding sulfide, the term “thio”refers to the sulfide, and the term “sulfonyl” refers to the fullyoxidized S(O)₂ moiety.

“aroyl”—a C(O)Ar, wherein Ar is as phenyl, naphthyl, or aryl alkylderivative such as defined above, such group include but are notelimited to benzyl and phenethyl.

“alkanoyl”—a C(O)C₁₋₁₀ alkyl wherein the alkyl is as defined above.

It is recognized that the compounds of the present invention may existas stereoisomers, regioisomers, or diastereiomers. These compounds maycontain one or more asymmetric carbon atoms and may exist in racemic andoptically active forms. All of these compounds are included within thescope of the present invention.

Synthetic Methods

The compounds of Formula (I) may be obtained by applying syntheticprocedures, some of which are illustrated in Scheme I below. Thesynthesis provided for in these Schemes is applicable for producingcompounds of Formula (I) having a variety of different R₁, R₂, and R₄groups which are reacted, employing optional substituents which aresuitably protected, to achieve compatibility with the reactions outlinedherein. Subsequent deprotection, in those cases, then affords compoundsof the nature generally disclosed. Once the nucleus has beenestablished, further compounds of Formula (I) may be prepared byapplying standard techniques for functional group interconversion allwell known in this art.

Compounds of Formula (I) are cycloalkylene derivatives which may bereadily prepared using procedures well known to those of skill in theart and may be prepared by analogous methods to those indicated hereinbelow.

Suitable protecting groups for use in the present invention, are wellknown in the art and described in many references, for instance,Protecting Groups in Organic Synthesis, Greene T W, Wiley-Interscience,New York, 1981.

Pharmaceutically acid addition salts of compounds of formula (I) may beobtained in known manner, for example by treatment thereof with anappropriate amount of acid in the presence of a suitable solvent.

The invention will now be described by reference to the followingexamples which are merely illustrative and are not to be construed as alimitation of the scope of the present invention.

Synthetic Examples EXAMPLE 11-[4-(2-Aminopyrimidinyl)]-2-(4-fluorophenyl)cyclopenten-1-ene (a)1-Carboethoxy-2-(trifluoromethylsulfonyloxy)cyclopentene

To a solution of 2-ethoxycarbonyl-1-cyclopentanone (5.0 mL, 33.3 mMol)in dichloromethane (40 mL) was added sodium hydride (1.8 g.; 60%, 43.3mMol.) and the mixture was stirred for 10 min. The mixture was cooled to−30° C. and trifluoromethanesulfonic anhydride (6.0 mL, 40 mMol.) wasadded the mixture was warmed to 0° C. over an hour and was diluted withwater and ether. The organic phase was separated and washed with brine.Evaporation of the solvent yielded the triflate as a colorless oil (9.2g. 96%)

(b) Ethyl 2-(4-fluorophenyl)cyclopentene-1-carboxylate

To a solution of the triflate from previous example (5.0 g, 20 mMol.)(40 mL) was added tetrakis(triphenyl)palladium(O), 4-fluorophenylboronicacid (2.9 g., 20 mMol.) and sodium carbonate (2.3 g., 22.2 mMol in 50 mLof water). The mixture was refluxed for about 1 hour and cooled. Thereaction mixture was diluted with ether and washed with brineevaporation of the solvent and flash chromatography afforded the titlecompound as a colorless oil (4.3 g. 92%).

N,O-Dimethyl 2-(4-fluorophenyl)cyclopentene-1-hydroxamate

To a solution of the ester (4.0 g, 17.1 mMol.) from the previous examplein toluene (80 mL) was added a solution of methyl chloroaluminumN,O-dimethyl-hydroxamide (20 mMol.) in toluene (70 mL) and the mixturewas stirred at 80° C. for 12 h. Quenched with 3N HCl. and extracted withEtOAc (3*50 mL.). Evaporation of the solvent and flash chromatographyafforded 1.1 g. of amide (25%)

1-Acetyl 2-(4-fluorophenyl)cyclopent-1-ene

To a solution of the amide (1.0 g., 4.0 mMol.) from previous step inanhydrous THF (20 mL) at −78° C. was added a 3M methyl magnesium bromide(12 mL, 37 mMol.) and the mixture was gradually warmed to roomtemperature over 2 h. The mixture was quenched with Sat. NH₄Cl andextracted with EtOAc. Evaporation of the solvent afforded themethylketone as a yellow oil 0.9 g (95%). ¹HNMR (CDCl₃) δ7.25 (q, 2H),7.05 (t, 2H), 2.73 (m, 4H), 2.0 (s, 3H), 1.92 (m, 5H).

1-[(3-Dimethylamino)propenoyl] 2-(4-fluorophenyl)cyclopent-1-ene

To a solution of the methyl ketone from the previous step (0.9 g., 4.0mMol) in DMF (27 mL.) was added tert-butoxy-bis(dimethylamino)-methane(Bredrick's reagent, 3 mL, 14.7 mMol.) and the mixture was stirred at110° C. for 4 h. The mixture was cooled to room temperature and dilutedwith water and The organic extract was separated and washed with brineevaporation of the solvent and flash chromatography yielded the titlecompound as dark oil (1.038 g., 98%). ¹HNMR (CDCl₃) δ7.90 (s,1H), 7.30(q, 2H), 6.93 (t, 2H), 4.86 (d, 1H), 2.93 (s, 3H), 2.84 (s, 3H), 2.80(m, 4H), 1.95 (m, 2H).

1-[4-(2-Aminopyrimidinyl)]-2-(4-fluorophenyl)cyclopenten-1-ene

To a solution of sodium metal (0.92 g., 4 mMol.) in EtOH (3.0 mL) wasadded guanidinehydrochloride (0.31 g.) and the mixture was stirred for10 min. Enamine from the previous example (1.03 mMol., 4.0 mMol.) inEtOH (30 mL) was added to the above solution and the mixture wasrefluxed overnight. The mixture was cooled and concentrated. The residuewas taken u in EtOAc and the salts were filtered off. Evaporation of thesolvent and crystallization from EtOH/H₂O afforded the requirescyclopentene as yellow plates. mp 134-135° C. ¹HNMR (CDCl₃) δ(0.25 g.,25%) 8.02(d, 1H), 7.17 (q, 2H), 6.96 (t, 2H), 6.27 (d, 1h), 5.05(broads, 2H), 2.94 (m, 4H), 2.06 (m, 2H). MS (ESP+, m/z) 256.1.

Methods of Treatment

The compounds of Formula (I) or a pharmaceutically acceptable saltthereof can be used in the manufacture of a medicament for theprophylactic or therapeutic treatment of any disease state in a human,or other mammal, which is exacerbated or caused by excessive orunregulated cytokine production by such mammal's cell, such as but notlimited to monocytes and/or macrophages.

As used herein, unless specifically indicated, compounds of Formula (I)also include compounds of Formula (II).

Compounds of Formula (I) are capable of inhibiting proinflammatorycytokines, such as IL-1, IL-6, IL-8 and TNF and are therefore of use intherapy. IL-1, IL-6, IL-8 and TNF affect a wide variety of cells andtissues and these cytokines, as well as other leukocyte-derivedcytokines, are important and critical inflammatory mediators of a widevariety of disease states and conditions. The inhibition of thesepro-inflammatory cytokines is of benefit in controlling, reducing andalleviating many of these disease states.

Accordingly, the present invention provides a method of treating acytokine-mediated disease which comprises administering an effectivecytokine-interfering amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof.

In particular, compounds of Formula (I) or a pharmaceutically acceptablesalt thereof are of use in the prophylaxis or therapy of any diseasestate in a human, or other mammal, which is exacerbated by or caused byexcessive or unregulated IL-1, IL-8 or TNF production by such mammal'scell, such as, but not limited to, monocytes and/or macrophages.

Accordingly, in another aspect, this invention relates to a method ofinhibiting the production of IL-1 in a mammal in need thereof whichcomprises administering to said mammal an effective amount of a compoundof Formula (I) or a pharmaceutically acceptable salt thereof.

There are many disease states in which excessive or unregulated IL-1production is implicated in exacerbating and/or causing the disease.These include rheumatoid arthritis, osteoarthritis, stroke, endotoxemiaand/or toxic shock syndrome, other acute or chronic inflammatory diseasestates such as the inflammatory reaction induced by endotoxin orinflammatory bowel disease, tuberculosis, atherosclerosis, muscledegeneration, multiple sclerosis, cachexia, bone resorption, psoriaticarthritis, Reiter's syndrome, rheumatoid arthritis, gout, traumaticarthritis, rubella arthritis and acute synovitis. Recent evidence alsolinks IL-1 activity to diabetes, pancreatic β cells and Alzheimer'sdisease.

In a further aspect, this invention relates to a method of inhibitingthe production of TNF in a mammal in need thereof which comprisesadministering to said mammal an effective amount of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof.

Excessive or unregulated TNF production has been implicated in mediatingor exacerbating a number of diseases including rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, stroke, cerebral malaria, chronic pulmonary inflammatorydisease, silicosis, pulmonary sarcoisosis, bone resorption diseases,such as osteoporosis, reperfusion injury, graft vs. host reaction,allograft rejections, fever and myalgias due to infection, such asinfluenza, cachexia secondary to infection or malignancy, cachexiasecondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDSrelated complex), keloid formation, scar tissue formation, inflammatorybowel disease, Crohn's disease, ulcerative colitis and pyresis.

Compounds of Formula (I) are also useful in the treatment of viralinfections, where such viruses are sensitive to upregulation by TNF orwill elicit TNF production in vivo. The viruses contemplated fortreatment herein are those that produce TNF as a result of infection, orthose which are sensitive to inhibition, such as by decreasedreplication, directly or indirectly, by the TNF inhibiting-compounds ofFormula (1). Such viruses include, but are not limited to HIV-1, HIV-2and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpesgroup of viruses, such as but not limited to, Herpes Zoster and HerpesSimplex. Accordingly, in a further aspect, this invention relates to amethod of treating a mammal afflicted with a human immunodeficiencyvirus (HIV) which comprises administering to such mammal an effectiveTNF inhibiting amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof.

Compounds of Formula (I) may also be used in association with theveterinary treatment of mammals, other than in humans, in need ofinhibition of TNF production. TNF mediated diseases for treatment,therapeutically or prophylactically, in animals include disease statessuch as those noted above, but in particular viral infections. Examplesof such viruses include, but are not limited to, lentivirus infectionssuch as, equine infectious anaemia virus, caprine arthritis virus, visnavirus, or maedi virus or retrovirus infections, such as but not limitedto feline immunodeficiency virus (FIV), bovine immunodeficiency virus,or canine immunodeficiency virus or other retroviral infections.

The compounds of Formula (I) may also be used topically in the treatmentor prophylaxis of topical disease states mediated by or exacerbated byexcessive cytokine production, such as by IL-1 or TNF respectively, suchas inflamed joints, eczema, contact dermititis, psoriasis and otherinflammatory skin conditions such as sunburn; inflammatory eyeconditions including conjunctivitis; pyresis, pain and other conditionsassociated with inflammation.

Compounds of Formula (I) have also been shown to inhibit the productionof IL-8 (Interleukin-8, NAP). Accordingly, in a further aspect, thisinvention relates to a method of inhibiting the production of IL-8 in amammal in need thereof which comprises administering to said mammal aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof.

There are many disease states in which excessive or unregulated IL-8production is implicated in exacerbating and/or causing the disease.These diseases are characterized by massive neutrophil infiltration suchas, psoriasis, inflammatory bowel disease, asthma, cardiac and renalreperfusion injury, adult respiratory distress syndrome, thrombosis andglomerulonephritis. All of these diseases are associated with increasedIL-8 production which is responsible for the chemotaxis of neutrophilsinto the inflammatory site. In contrast to other inflammatory cytokines(IL-1, TNF, and IL-6), IL-8 has the unique property of promotingneutrophil chemotaxis and activation. Therefore, the inhibition of IL-8production would lead to a direct reduction in the neutrophilinfiltration.

The compounds of Formula (I) are administered in an amount sufficient toinhibit cytokine, in particular IL-1, IL-6, IL-8 or TNF, production suchthat it is regulated down to normal levels, or in some case to subnormallevels, so as to ameliorate or prevent the disease state. Abnormallevels of IL-1, IL-6, IL-8 or TNF, for instance in the context of thepresent invention, constitute: (i) levels of free (not cell bound) IL-1,IL-6, IL-8 or TNF greater than or equal to 1 picogram per ml; (ii) anycell associated IL-1, IL-6, IL-8 or TNF; or (iii) the presence of IL-1,IL-6, IL-8 or TNF mRNA above basal levels in cells or tissues in whichIL-1, IL-6, IL-8 or TNF, respectively, is produced.

The discovery that the compounds of Formula (I) are inhibitors ofcytokines, specifically IL-1, IL-6, IL-8 and TNF is based upon theeffects of the compounds of Formulas (I) on the production of the IL-1,IL-8 and TNF in in vitro assays which are described herein.

As used herein, the term “inhibiting the production of IL-1 (IL-6, IL-8or TNF)” refers to:

a) a decrease of excessive in vivo levels of the cytokine (IL-1, IL-6,IL-8 or TNF) in a human to normal or sub-normal levels by inhibition ofthe in vivo release of the cytokine by all cells, including but notlimited to monocytes or macrophages;

b) a down regulation, at the genomic level, of excessive in vivo levelsof the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal orsub-normal levels;

c) a down regulation, by inhibition of the direct synthesis of thecytokine (IL-1, IL-6, IL-8 or TNF) as a postranslational event; or

d) a down regulation, at the translational level, of excessive in vivolevels of the cytokine (IL-1, IL-6, IL-8 or TNF) in a human to normal orsub-normal levels.

As used herein, the term “TNF mediated disease or disease state” refersto any and all disease states in which TNF plays a role, either byproduction of TNF itself, or by TNF causing another monokine to bereleased, such as but not limited to IL-1, IL-6 or IL-8. A disease statein which, for instance, IL-1 is a major component, and whose productionor action, is exacerbated or secreted in response to TNF, wouldtherefore be considered a disease stated mediated by TNF.

As used herein, the term “cytokine” refers to any secreted polypeptidethat affects the functions of cells and is a molecule which modulatesinteractions between cells in the immune, inflammatory or hematopoieticresponse. A cytokine includes, but is not limited to, monokines andlymphokines, regardless of which cells produce them. For instance, amonokine is generally referred to as being produced and secreted by amononuclear cell, such as a macrophage and/or monocyte. Many other cellshowever also produce monokines, such as natural killer cells,fibroblasts, basophils, neutrophils, endothelial cells, brainastrocytes, bone marrow stromal cells, epideral keratinocytes andB-lymphocytes. Lymphokines are generally referred to as being producedby lymphocyte cells. Examples of cytokines include, but are not limitedto, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8),Tumor Necrosis Factor-alpha (TNF-α) and Tumor Necrosis Factor beta(TNF-β).

As used herein, the term “cytokine interfering” or “cytokine suppressiveamount” refers to an effective amount of a compound of Formula (I) whichwill cause a decrease in the in vivo levels of the cytokine to normal orsub-normal levels, when given to a patient for the prophylaxis ortreatment of a disease state which is exacerbated by, or caused by,excessive or unregulated cytokine production.

As used herein, the cytokine referred to in the phrase “inhibition of acytokine, for use in the treatment of a HIV-infected human” is acytokine which is implicated in (a) the initiation and/or maintenance ofT cell activation and/or activated T cell-mediated HIV gene expressionand/or replication and/or (b) any cytokine-mediated disease associatedproblem such as cachexia or muscle degeneration.

As TNF-β (also known as lymphotoxin) has close structural homology withTNF-α (also known as cachectin) and since each induces similar biologicresponses and binds to the same cellular receptor, both TNF-α and TNF-βare inhibited by the compounds of the present invention and thus areherein referred to collectively as “TNF” unless specifically delineatedotherwise.

A new member of the MAP kinase family, alternatively termed CSBP, p38,or RK, has been identified independently by several laboratoriesrecently [See Lee et al., Nature, Vol. 300 n(72), 739-746 (1994)].Activation of this novel protein kinase via dual phosphorylation hasbeen observed in different cell systems upon stimulation by a widespectrum of stimuli, such as physicochemical stress and treatment withipopolysaccharide or proinflammatory cytokines such as interleukin-1 andtumor necrosis factor. The cytokine biosynthesis inhibitors, of thepresent invention, compounds of Formula (I), have been determined to bepotent and selective inhibitors of CSBP/p38/RK kinase activity. Theseinhibitors are of aid in determining the signaling pathways involvementin inflammatory responses. In particular, for the first time adefinitive signal transduction pathway can be prescribed to the actionof lipopolysaccharide in cytokine production in macrophages. In additionto those diseases already noted, treatment of stroke, neurotrauma,cardiac and renal reperfusion injury, thrombosis, glomerulonephritis,diabetes and pancreatic β cells, multiple sclerosis, muscledegeneration, eczema, psoriasis, sunburn, and conjunctivitis are alsoincluded.

The cytokine inhibitors were subsequently tested in a number of animalmodels for anti-inflammatory activity. Model systems were chosen thatwere relatively insensitive to cyclooxygenase inhibitors in order toreveal the unique activities of cytokine suppressive agents. Theinhibitors exhibited significant activity in many such in vivo studies.Most notable are its effectiveness in the collagen-induced arthritismodel and inhibition of TNF production in the endotoxic shock model. Inthe latter study, the reduction in plasma level of TNF correlated withsurvival and protection from endotoxic shock related mortality. Also ofgreat importance are the compounds effectiveness in inhibiting boneresorption in a rat fetal long bone organ culture system. Griswold etal., (1988) Arthritis Rheum. 31:1406-1412; Badger, et al., (1989) Circ.Shock 27, 51-61; Votta et al., (1994)in vitro. Bone 15, 533-538; Lee etal., (1993). B Ann. N. Y. Acad Sci. 696, 149-170.

Another aspect of the present invention is to the novel use of theseCSBP/cytokine inhibitors for the treatment of chronic inflammatory orproliferative or angiogenic diseases which are caused by excessive, orinappropriate angiogenesis.

Chronic diseases which have an inappropriate angiogenic component arevarious ocular neovasularizations, such as diabetic retinopathy andmacular degeneration. Other chronic diseases which have an excessive orincreased proliferation of vasculature are tumor growth and metastasis,atherosclerosis, and certain arthritic conditions. Therefore cytokineinhibitors will be of utility in the blocking of the angiogeniccomponent of these disease states.

The term “excessive or increased proliferation of vasculatureinappropriate angiogenesis” as used herein includes, but is not limitedto, diseases which are characterized by hemangiomas and ocular diseases.

The term “inappropriate angiogenesis” as used herein includes, but isnot limited to, diseases which are characterized by vesicleproliferation with accompanying tissue proliferation, such as occurs incancer, metastasis, arthritis and atherosclerosis.

In order to use a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in therapy, it will normally be Formulated intoa pharmaceutical composition in accordance with standard pharmaceuticalpractice. This invention, therefore, also relates to a pharmaceuticalcomposition comprising an effective, non-toxic amount of a compound ofFormula (I) and a pharmaceutically acceptable carrier or diluent.

Compounds of Formula (I), pharmaceutically acceptable salts thereof andpharmaceutical compositions incorporating such may conveniently beadministered by any of the routes conventionally used for drugadministration, for instance, orally, topically, parenterally or byinhalation. The compounds of Formula (I) may be administered inconventional dosage forms prepared by combining a compound of Formula(I) with standard pharmaceutical carriers according to conventionalprocedures. The compounds of Formula (I) may also be administered inconventional dosages in combination with a known, second therapeuticallyactive compound. These procedures may involve mixing, granulating andcompressing or dissolving the ingredients as appropriate to the desiredpreparation. It will be appreciated that the form and character of thepharmaceutically acceptable character or diluent is dictated by theamount of active ingredient with which it is to be combined, the routeof administration and other well-known variables. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The pharmaceutical carrier employed may be, for example, either a solidor liquid. Exemplary of solid carriers are lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. Exemplary of liquid carriers are syrup, peanut oil, oliveoil, water and the like. Similarly, the carrier or diluent may includetime delay material well known to the art, such as glycerylmono-stearate or glyceryl distearate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg. to about 1 g. When a liquid carrier is used,the preparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampule or nonaqueousliquid suspension.

Compounds of Formula (I) may be administered topically, that is bynon-systemic administration. This includes the application of a compoundof Formula (I) externally to the epidermis or the buccal cavity and theinstillation of such a compound into the ear, eye and nose, such thatthe compound does not significantly enter the blood stream. In contrast,systemic administration refers to oral, intravenous, intraperitoneal andintramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as liniments, lotions, creams, ointmentsor pastes, and drops suitable for administration to the eye, ear ornose. The active ingredient may comprise, for topical administration,from 0.001% to 10% w/w, for instance from 1% to 2% by weight of theformulation. It may however comprise as much as 10% w/w but preferablywill comprise less than 5% w/w, more preferably from 0.1% to 1% w/w ofthe formulation.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy base. The base may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives or a fattyacid such as steric or oleic acid together with an alcohol such aspropylene glycol or a macrogel. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurfactant such as a sorbitan ester or a polyoxyethylene derivativethereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at98-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Compounds of formula (I) may be administered parenterally, that is byintravenous, intramuscular, subcutaneous intranasal, intrarectal,intravaginal or intraperitoneal administration. The subcutaneous andintramuscular forms of parenteral administration are generallypreferred. Appropriate dosage forms for such administration may beprepared by conventional techniques. Compounds of Formula (I) may alsobe administered by inhalation, that is by intranasal and oral inhalationadministration. Appropriate dosage forms for such administration, suchas an aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques.

For all methods of use disclosed herein for the compounds of Formula(I), the daily oral dosage regimen will preferably be from about 0.1 toabout 80 mg/kg of total body weight, preferably from about 0.2 to 30mg/kg, more preferably from about 0.5 mg to 15 mg. The daily parenteraldosage regimen about 0.1 to about 80 mg/kg of total body weight,preferably from about 0.2 to about 30 mg/kg, and more preferably fromabout 0.5 mg to 15 mg/kg. The daily topical dosage regimen willpreferably be from 0.1 mg to 150 mg, administered one to four,preferably two or three times daily. The daily inhalation dosage regimenwill preferably be from about 0.01 mg/kg to about 1 mg/kg per day. Itwill also be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a compound of Formula (I)or a pharmaceutically acceptable salt thereof will be determined by thenature and extent of the condition being treated, the form, route andsite of administration, and the particular patient being treated, andthat such optimums can be determined by conventional techniques. It willalso be appreciated by one of skill in the art that the optimal courseof treatment, i.e., the number of doses of a compound of Formula (I) ora pharmaceutically acceptable salt thereof given per day for a definednumber of days, can be ascertained by those skilled in the art usingconventional course of treatment determination tests.

The invention will now be described by reference to the followingbiological examples which are merely illustrative and are not to beconstrued as a limitation of the scope of the present invention.

Biological Examples

The cytokine-inhibiting effects of compounds of the present inventionwere determined by the following in vitro assays:

Assays for Interleukin-1 (IL-1), Interleukin-8 (IL-8 ), and TumourNecrosis Factor (TNF) are well known in the art, and may be found in anumber of publications, and patents. Representative suitable assays foruse herein are described in Adams et al., U.S. Pat. No. 5,593,992, whosedisclosure is incorporated by reference in its entirety.

Interleukin-1 (IL-1)

Human peripheral blood monocytes are isolated and purified from eitherfresh blood preparations from volunteer donors, or from blood bank buffycoats, according to the procedure of Colotta et al, J Immunol, 132, 936(1984). These monocytes (1×10⁶) are plated in 24-well plates at aconcentration of 1-2 million/ml per well. The cells are allowed toadhere for 2 hours, after which time non-adherent cells are removed bygentle washing. Test compounds are then added to the cells for 1 hbefore the addition of lipopolysaccharide (50 ng/ml), and the culturesare incubated at 37° C. for an additional 24 h. At the end of thisperiod, culture supernatants are removed and clarified of cells and alldebris. Culture supernatants are then immediately assayed for IL-1biological activity, either by the method of Simon et al., J. Immunol.Methods, 84, 85, (1985) (based on ability of IL-1 to stimulate aInterleukin 2 producing cell line (EL-4) to secrete IL-2, in concertwith A23187 ionophore) or the method of Lee et al., J. ImmunoTherapy, 6(1), 1-12 (1990) (ELISA assay).

In vivo TNF Assay

(1) Griswold et al., Drugs Under Exp. and Clinical Res.,XIX (6), 243-248(1993); or

(2) Boehm, et al., Journal Of Medicinal Chemistry 39, 3929-3937 (1996)whose disclosures are incorporated by reference herein in theirentirety.

LPS-induced TNFα Production in Mice and Rats

In order to evaluate in vivo inhibition of LPS-induced TNFα productionin rodents, both mice and rats are injected with LPS.

Mouse Method

Male Balb/c mice from Charles River Laboratories are pretreated (30minutes) with compound or vehicle. After the 30 min. pretreat time, themice are given LPS (lipopolysaccharide from Esherichia coli Serotype055-85, Sigma Chemical Co., St Louis, Mo.) 25 ug/mouse in 25 ulphosphate buffered saline (pH 7.0) intraperitoneally. Two hours laterthe mice are killed by CO₂ inhalation and blood samples are collected byexsanguination into heparinized blood collection tubes and stored onice. The blood samples are centrifuged and the plasma collected andstored at −20° C. until assayed for TNFα by ELISA.

Rat Method

Male Lewis rats from Charles River Laboratories are pretreated atvarious times with compound or vehicle. After a determined pretreattime, the rats are given LPS (lipopolysaccharide from Esherichia coliSerotype 055-85, Sigma Chemical Co., St Louis, Mo.) 3.0 mg/kgintraperitoneally. The rats are killed by CO₂ inhalation and heparinizedwhole blood is collected from each rat by cardiac puncture 90 minutesafter the LPS injection. The blood samples are centrifuged and theplasma collected for analysis by ELISA for TNFα levels.

ELISA Method

TNFα levels were measured using a sandwich ELISA, as described inOlivera et al., Circ. Shock, 37, 301-306, (1992), whose disclosure isincorporated by reference in its entirety herein, using a hamstermonoclonal antimurine TNFα (Genzyme, Boston, Mass.) as the captureantibody and a polyclonal rabbit antimurine TNFα (Genzyme) as the secondantibody. For detection, a peroxidase-conjugated goat antirabbitantibody (Pierce, Rockford, Ill.) was added, followed by a substrate forperoxidase (1 mg/ml orthophenylenediamine with 1% urea peroxide). TNFαlevels in the plasma samples from each animal were calculated from astandard curve generated with recombinant murine TNFα (Genzyme).

LPS-Stimulated Cytokine Production in Human Whole Blood

Assay: Test compound concentrations were prepared at 10×concentrationsand LPS prepared at 1 ug/mI (final conc. of 50 ng/ml LPS) and added in50 uL volumes to 1.5 mL eppendorf tubes. Heparinized human whole bloodwas obtained from healthy volunteers and was dispensed into eppendorftubes containing compounds and LPS in 0.4 mL volumes and the tubesincubated at 37 C. Following a 4 hour incubation, the tubes werecentrifuged at 5000 rpm for 5 minutes in a TOMY microfuge, plasma waswithdrawn and frozen at −80 C.

Cytokine measurement: IL-I and/or TNF were quantified using astandardized ELISA technology. An in-house ELISA kit was used to detecthuman IL-1 and TNF. Concentrations of IL-1 or TNF were determined fromstandard curves of the appropriate cytokine and IC50 values for testcompound (concentration that inhibited 50% of LPS-stimulated cytokineproduction) were calculated by linear regression analysis.

Prostoglandin Endoperoxide Synthase-2 (PGHS-2) Assay

This assay describes a method for determining the inhibitory effects ofcompounds of Formula (I) on human PGHS-2 protein expression in LPSstimulated human monocytes. A suitable assay for PGHS-2 proteinexpression may be found in a number of publications, including U.S. Pat.No. 5,593,992 whose disclosure is incorporated herein by reference.

Cytokine Specific Binding Protein Assay

A radiocompetitive binding assay was developed to provide a highlyreproducible primary screen for structure-activity studies. This assayprovides many advantages over the conventional bioassays which utilizefreshly isolated human monocytes as a source of cytokines and ELISAassays to quantify them. Besides being a much more facile assay, thebinding assay has been extensively validated to highly correlate withthe results of the bioassay. A specific and reproducible cytokineinhibitor binding assay was developed using soluble cystosolic fractionfrom THP.1 cells and a radiolabeled compound. Patent Application U.S.Ser. No. 08/123175 Lee et al., filed September 1993, USSN; Lee et al.,PCT 94/10529 filed Sep. 16, 1994 and Lee et al., Nature 300, n(72),739-746 (December 1994) whose disclosures are incorporated by referenceherein in its entirety describes the above noted method for screeningdrugs to identify compounds which interact with and bind to the cytokinespecific binding protein (hereinafter CSBP). However, for purposesherein the binding protein may be in isolated form in solution, or inimmobilized form, or may be genetically engineered to be expressed onthe surface of recombinant host cells such as in phage display system oras fusion proteins. Alternatively, whole cells or cytosolic fractionscomprising the CSBP may be employed in the screening protocol.Regardless of the form of the binding protein, a plurality of compoundsare contacted with the binding protein under conditions sufficient toform a compound/binding protein complex and compound capable of forming,enhancing or interfering with said complexes are detected.

CSBP Kinase Assay

This assay measures the CSBP-catalyzed transfer of ³²P from [a-³²P]ATPto threonine residue in an epidermal growth factor receptor(EGFR)-derived peptide (T669) with the following sequence:KRELVEPLTPSGEAPNQALLR (residues 661-681). (See Gallagher et al.,“Regulation of Stress Induced Cytokine Production by PyridinylImidazoles: Inhibition of CSPB Kinase”, BioOrganic & MedicinalChemistry, to be published 1996).

Kinase reactions (total volume 30 ul) contain: 25 mM Hepes buffer, pH7.5; 10 mM MgCl₂; 170 uM ATP⁽¹⁾; 10 uM Na ortho vanadate; 0.4 mM T669peptide; and 20-80 ng of yeast-expressed purified CSBP2 (see Lee et al.,Nature 300, n(72), 739-746 (December 1994)). Compounds (5 ul from [6×]stock⁽²⁾) are pre-incubated with the enzyme and peptide for 20 min onice prior to starting the reactions with 32P/MgATP. Reactions areincubated at 30° C. for 10 min and stopped by adding 10 ul of 0.3 Mphosphoric acid. 32P-labeled peptide is separated on phosphocellulose(Wattman, p81) filters by spotting 30 ul reaction mixture. Filters arewashed 3 times with 75 mM phosphoric acid followed by 2 washes with H₂O,and counted for 32P.

(1) The Km of CSBP for ATP was determined to be 170 uM. Therefore,compounds screened at the Km value of ATP.

(2) Compounds are usually dissolved in DMSO and are diluted in 25 mMHepes buffer to get final concentration of DMSO of 0.17%.

Representative compounds of Formula (I), Example 1 demonstrated apositive inhibitory activity in this assay having an IC50<50 uM.

TNF-a in Traumatic Brain Injury Assay

This assay provides for examination of the expression of tumor necrosisfactor mRNA in specific brain regions which follow experimentallyinduced lateral fluid-percussion traumatic brain injury (TBI) in rats.Since TNF-a is able to induce nerve growth factor (NGF) and stimulatethe release of other cytokines from activated astrocytes, thispost-traumatic alteration in gene expression of TNF-a plays an importantrole in both the acute and regenerative response to CNS trauma. Asuitable assay may be found in WO 97/35856 whose disclosure isincorporated herein by reference.

CNS Injury Model for IL-b mRNA

This assay characterizes the regional expression of interleukin-1β(IL-1β) mRNA in specific brain regions following experimental lateralfluid-percussion traumatic brain injury (TBI) in rats Results from theseassays indicate that following TBI, the temporal expression of IL-1βmRNA is regionally stimulated in specific brain regions. These regionalchanges in cytokines, such as IL-1β play a role in the post-traumaticpathologic or regenerative sequelae of brain injury. A suitable assaymay be found in WO 97/35856 whose disclosure is incorporated herein byreference.

Angiogenesis Assay

Described in WO 97/32583, whose disclosure is incorporated herein byreference, is an assay for determination of inflammatory angiogenesiswhich may be used to show that cytokine inhibition will stop the tissuedestruction of excessive or inappropriate proliferation of bloodvessels.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe are can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

What is claimed is:
 1. A compound of the formula

wherein R₁ is a 4-pyridyl, 4-pyrimidinyl, 4-pyridazinyl,1,2,4-triazin-5-yl, 4-quinolyl, 6-isoquinolinyl, or quinazolin-4-ylring, which ring is optionally substituted independently one to threetimes with Y, NHR_(a), optionally substituted C₁₋₄ alkyl, halogen,hydroxyl, optionally substituted C₁₋₄ alkoxy, optionally substitutedC₁₋₄ alkylthio, optionally substituted C₁₋₄ alkylsulfinyl, CH₂OR₁₂,amino, mono and di-C₁₋₆ alkyl substituted amino, N(R₁₀)C(O)R_(b),N(R₁₀)S(O)₂R_(d), or an N-heterocyclyl ring which ring has from 5 to 7members and optionally contains an additional heteroatom selected fromoxygen, sulfur or NR₁₅; Y is X₁—R_(a); X₁ is sulfur or oxygen; R_(a) isC₁₋₆ alkyl, aryl, arylC₁₋₆ alkyl, heterocyclic, heterocyclylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl moiety, wherein each of thesemoieties may be optionally substituted; R_(b) is hydrogen, C₁₋₆ alkyl,C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl, heteroarylC₁₋₄ alkyl,heterocyclyl, or heterocyclylC₁₋₄ alkyl; R_(d) is C₁₋₆ alkyl, C₃₋₇cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl, heteroarylC₁₋₄ alkyl,heterocyclyl, or heterocyclylC₁₋₄ alkyl; Rg is independently hydrogen orR₂; n is 0, or an integer having a value of 1 to 10; v is 0, or aninteger having a value of 1 or 2; m is 0, or the integer having a valueof 1 or 2; m′ is an integer having a value of 1 or 2, m″ is 0, or aninteger having a value of 1 to 5; s is an integer having a value of 1 to5; t is 0, or an integer having a value of 1, 2 or 3; R₂ isindependently hydrogen, (CR₁₀R₂₃)_(n)OR₉, (CR₁₀R₂₃)_(n)OR₁₁, C₁₋₁₀alkyl, halo-substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₇cycloalkyl, C₃₋₇cycloalkylC₁₋₁₀ alkyl, C₅₋₇ cycloalkenyl, C₅₋₇cycloalkenyl C₁₋₁₀ alkyl, aryl, arylC₁₋₁₀ alkyl, heteroaryl,heteroarylC₁₋₁₀ alkyl, heterocyclyl, heterocyclylC₁₋₁₀ alkyl,(CR₁₀R₂₃)_(n)S(O)_(m)R₁₈, (CR₁₀R₂₃)_(n)NHS(O)₂R₁₈, (CR₁₀R₂₃)_(n)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)NO₂, (CR₁₀R₂₃)_(n)CN, (CR₁₀R₂₃)_(n) S(O)m′NR₁₃R₁₄,(CR₁₀R₂₃)_(n)C(Z)R₁₁, (CR₁₀R₂₃)_(n)OC(Z)R₁₁(CR₁₀R₂₃)_(n)C(Z)OR₁₁,(CR₁₀R₂₃)_(n)C(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)C(Z)NR₁₁OR₉,(CR₁₀R₂₃)_(n)NR₁₀C(Z)R₁₁, (CR₁₀R₂₃)_(n)NR10C(Z)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)N(OR₆)C(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)N(OR6)C(Z)R₁₁,(CR₁₀R₂₃)_(n)C(═NOR₆)R₁₁, (CR₁₀R₂₃)_(n)NR₁₀C(═NR₁₉)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)OC(Z)NR₁₃R₁₄, (CR₁₀R₂₃)_(n)NR₁₀C(Z)NR₁₃R₁₄,(CR₁₀R₂₃)_(n)NR₁₀C(Z)OR₁₀, 5-(R₁₈)-1,2,4-oxadizaol-3-yl or4-(R₁₂)-5-(R₁₈R₁₉)-4,5-dihydro-1,2,4-oxadiazol-3-yl; wherein thecycloalkyl, cycloalkyl alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic and heterocyclic alkyl may be optionallysubstituted; R₃ is heterocyclyl, heterocyclylC₁₋₁₀ alkyl or R₈; R₄ isphenyl, naphth-1-yl or naphth-2-yl, or heteroaryl, which is optionallysubstituted by one to three substituents, each of which is independentlyselected, and which, for a 4-phenyl, 4-naphth-1-yl, 5-naphth-2-yl or6-naphth-2-yl substituent, is halogen, cyano, nitro, C(Z)NR₇R₁₇,C(Z)OR₁₆, (CR₁₀R₂₀)vCOR₁₂, SR₅, S(O)R₅, OR₁₂, halo-substituted-C₁₋₄alkyl, C₁₋₄ alkyl, ZC(Z)R₁₂, NR₁₀C(Z)R₁₆, or (CR₁₀R₂₀)vNR₁₀R₂₀ andwhich, for other positions of substitution, is halogen, cyano, nitro,phenyl, C(Z)NR₁₃R₁₄, C(Z)OR₃, (CR₁₀R₂₀)m″COR₃, S(O)mR₃, OR₃,halosubstituted-C₁₋₄ alkyl, C₁₋₁₀ alkyl, ZC(Z)R₃, optionally substitutedphenyl, (CR₁₀R₂₀)m″NR₁₀C(Z)R₃, NR₁₀S(O)m′R8, NR₁₀S(O)m′NR₇R₁₇, or(CR₁₀R₂₀)m″NR₁₃R₁₄; R₅ is hydrogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl or NR₇R₁₇, excluding the moieties SR₅ being SNR₇R₁₇ and SOR₅being SOH; R₆ is hydrogen, a pharmaceutically acceptable cation, C₁₋₁₀alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl, heteroarylC₁₋₄alkyl, heterocyclic, aroyl, or C₁₋₁₀ alkanoyl; R₇ and R₁₇ is eachindependently selected from hydrogen or C₁₋₄ alkyl or R₇ and R₁₇together with the nitrogen to which they are attached form aheterocyclic ring of 5 to 7 members which ring optionally contains anadditional heteroatom selected from oxygen, sulfur or NR₁₅; R₈ is C₁₋₁₀alkyl, halo-substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,C₃₋₇cycloalkyl, C₅₋₇ cycloalkenyl, aryl, arylC₁₋₁₀ alkyl, heteroaryl,heteroarylC₁₋₁₀ alkyl, (CR₁₀R₂₀)_(n)OR₁₁, (CR₁₀R₂₀)_(n)S(O)_(m)R₁₈,(CR₁₀R₂₀)_(n)NHS(O)₂R₁₈, or (CR₁₀R₂₀)_(n)NR₁₃R₁₄; wherein the aryl,arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted;R₉ is hydrogen, C(Z)R₁₁ or optionally substituted C₁₋₁₀ alkyl, S(O)₂R₁₈,optionally substituted aryl or optionally substituted aryl-C₁₋₄ alkyl;R₁₀ and R₂₀ is each independently selected from hydrogen or C₁₋₄ alkyl;R₁₁ is hydrogen, C₁₋₁₀ alkyl, C₃₋₇ cycloalkyl, heterocyclyl,heterocyclyl C₁₋₁₀ alkyl, aryl, arylC₁₋₁₀ alkyl, heteroaryl orheteroarylC₁₋₁₀ alkyl moiety, wherein the aryl, arylalkyl, heteroaryl,heteroaryl alkyl, heterocyclyl or heterocyclylalkyl moieties may beoptionally substituted; R₁₂ is hydrogen or R₁₆; R₁₃ and R₁₄ is eachindependently selected from hydrogen or optionally substituted C₁₋₄alkyl, optionally substituted aryl or optionally substituted arylC₁₋₄alkyl, or together with the nitrogen which they are attached form aheterocyclic ring of 5 to 7 members which ring optionally contains anadditional heteroatom selected from oxygen, sulfur or NR₉; R₁₅ ishydrogen, C₁₋₄ alkyl or C(Z)—C₁₋₄ alkyl; R₁₆ is C₁₋₄ alkyl,halo-substituted-C₁₋₄ alkyl, or C₃₋₇ cycloalkyl; R₁₈ is C₁₋₁₀ alkyl,C₃₋₇ cycloalkyl, heterocyclyl, aryl, arylC₁₋₁₀ alkyl, heterocyclyl,heterocyclyl-C₁₋₁₀ alkyl, heteroaryl or heteroarylalkyl moiety, whereinthe aryl, arylalkyl, heteroaryl, heteroaryl alkyl, heterocyclyl orheterocyclylalkyl moieties may be optionally substituted; R₁₉ ishydrogen, cyano, C₁₋₄ alkyl, C₃₋₇ cycloalkyl or aryl; R₂₃ is hydrogen,C₁₋₆ alkyl, C₃₋₇ cycloalkyl, aryl, arylC₁₋₄ alkyl, heteroaryl,heteroarylC₁₋₄ alkyl, heterocyclyl, or a heterocyclylC₁₋₄ alkyl moiety,all of moieties which may be optionally substituted; Z is oxygen orsulfur; or a pharmaceutically acceptable salt thereof.
 2. The compoundaccording to claim 1 wherein R₁ is an optionally substituted4-pyrimindyl, or 4-pyridyl.
 3. The compound according to claim 2 whereinthe substituent is Y, or NHR_(a).
 4. The compound according to claim 2wherein R₄ is an optionally substituted phenyl.
 5. The compoundaccording to claim 4 wherein the phenyl is substituted one or more timesindependently by halogen, SR₅, S(O)R₅, OR₁₂, halo-substituted-C₁₋₄alkyl, or C₁₋₄ alkyl.
 6. The compound according to claim 1 wherein tis
 1. 7. The compound according to claim 1 which is:1-[4-(2-Aminopyrinidinyl)]-2-(4-fluorophenyl)cyclopenten-1-ene, or apharmaceutically acceptable salt thereof.
 8. A pharmaceuticalcomposition comprising a compound according to claim 1 and apharmaceutically acceptable carrier or diluent.
 9. A method of treatingan inflammatory component of a CSBP/RK/p38 kinase mediated disease, in amammal in need thereof, which comprises administering to said mammal aneffective amount of a compound of Formula (I) according to claim
 1. 10.The method according to claim 9 wherein the CSBP/RK/p38 kinase mediateddisease is psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis,gout, traumatic arthritis, rubella arthritis and acute synovitis,rheumatoid spondylitis, osteoarthritis, gouty arthritis, sepsis, septicshock, endotoxic shock, gram negative sepsis, toxic shock syndrome,Alzheimer's disease, stroke, neurotrauma, asthma, adult respiratorydistress syndrome, cerebral malaria, chronic pulmonary inflammatorydisease, silicosis, pulmonary sarcososis, bone resorption disease,osteoporosis, restenosis, cardiac and renal reperfusion injury,thrombosis, glomerularonephritis, diabetes, graft vs. host reaction,allograft rejection, inflammatory bowel disease, Crohn's disease,ulcerative colitis, multiple sclerosis, muscle degeneration, diabeticretinopathy, macular degeneration, eczema, contact dermatitis,psoriasis, sunburn, or conjunctivitis.